Abstract

Mutation frequencies (MnFs) of the lacI transgene and mutation rates (MRs) of the endogenous hprt gene were analyzed in two mammary carcinoma cell lines that we established from mammary carcinomas that had been induced by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in female lacI-transgenic rats. Using the lacI transgene, corrected MnF, which is the number of independent lacI mutations that occurred while 102 cells expanded into 107 cells and which reflect the dynamic increase of point mutations, was measured. The corrected MnFs in the two mammary carcinoma cell lines (59 × 10-6 and 72 × 10-6 mutations) were significantly higher than that in the primary culture of normal mammary epithelium (4.7 × 10-6). MRs of the hprt gene in the two mammary carcinoma cell lines (8.2 × 10-7 and 11 × 10-7 mutations/hprt/cell division) were also higher than the same control (1.4 × 10-7). A:T to C:G transversion was observed at significantly higher frequencies in the two cell lines (6 of 24 and 6 of 25 for lacI; 10 of 67 and 19 of 92 for hprt) than in the control (0 of 6 for lacI; 0 of 4 for hprt). Taking advantage of the lacI transgene, high frequencies of A:T to C:G transversion (6 of 38 and 8 of 33, respectively) was also confirmed in the primary carcinomas of the two cell lines, which indicated the presence of a common abnormality in the cell lines and in the primary carcinomas. Both the established cell lines and their primary carcinomas were negative for microsatellite instability, which is known to be caused mainly by mismatch repair insufficiency and to increase point mutations, and for p53 mutations. These findings showed that the two cell lines, and possibly their primary carcinomas, had increases in the MRs of point mutations attributable to a mechanism(s) different from mismatch repair insufficiency, and we would suggest that such a state be designated as single nucleotide instability (SNI).

INTRODUCTION

Genomic instability is expected to be involved in a wide variety of tumors so that multiple mutations are accumulated in a single cell and multistep carcinogenesis would be completed
(1, 2)
. Genomic instability can be classified into different categories by its main targets, such as subtle sequences, chromosomal number, translocations, or gene amplifications
(3)
. Among the subtle sequence instabilities, microsatellite instability has been the most extensively studied. It is mainly caused by an insufficiency of a mismatch repair system and is observed in a variety of human cancers. It is involved in multistep carcinogenesis by mutating repeat sequences in tumor-related genes, such as TGFβ-RII, BAX, and IGFIIR(3, 4)
, and also by increasing point mutations in non-repeat unique sequences
(5,
6,
7,
8)
.

Another type of subtle sequence instability is the one that mainly increases spontaneous point mutations with little effect on microsatellite mutation. Considering the wide role of point mutations in oncogene activation and tumor-suppressor gene inactivation, it is expected that this type of genomic instability is present in many types of cancers, especially in those that lack microsatellite instability. However, regarding analyses of the MR
3
, which is defined as the number of mutations per cell division and is important in assessing genomic instability accurately, there is only one report in which an increase of the point MR without microsatellite instability was demonstrated using the hprt marker gene
(5, 9)
. The paucity of previous reports is mainly attributable to the labor-intensive procedures to measure MRs
(3)
and to the uncertainty in extrapolating the results obtained in cell lines to primary tumors.

To overcome the uncertainty in the extrapolation, transgenic animals with a marker gene, such as the lacI or lacZ gene, provide good resources. BBRs carry a transgenic tandem repeat of ∼40 copies of λ phage, into which the lacI marker gene was inserted. The lacI gene can be recovered as a phage particle from the genomic DNA of any normal or tumor tissue, and lacI mutations that took place in vivo can be identified
(10, 11)
. Although MRs cannot be measured in primary tumors, mutation characteristics can be analyzed in primary tumors using the lacI transgene. The mutation characteristics observed in cell lines, if any, could be examined to see whether or not they are also present in the primary tumor from which the cell lines were established. If the same mutation characteristics are present in the primary tumors, it indicates that a molecular mechanism that caused changes in the MRs in the cell lines are also present in the primary tumors.

Mammary carcinomas induced by PhIP are known to show very rare events of microsatellite mutations
(12)
, as in the cases of most human breast cancers
(13)
. In this study, we established two cell lines from mammary carcinomas induced by oral feeding of PhIP to (BBR × Sprague Dawley)F1 rats. MnFs of the lacI transgene and MRs of the endogenous hprt gene were examined in the carcinoma cell lines. Mutation characteristics of the lacI transgene were analyzed in the cell lines and in the primary carcinomas from which they were established.

MATERIALS AND METHODS

Carcinogenicity Test, Cell Line Establishment, and Primary Culture.

BBRs were purchased from Stratagene (La Jolla, CA). As reported previously
(14)
, 33 (BBR × Sprague Dawley)F1 female rats were given 10 doses of PhIP (75 mg/kg/day PhIP-HCl) in water by gavage from the age of 6 weeks and were killed at the ages of 56–69 weeks. Most of the macroscopic mammary carcinomas were divided into three sections for histological examination, nucleic acid extraction, and primary culture. The section for primary culture was trimmed of surrounding connective tissue and cut into pieces of less than 1 mm3. Cells were grown in DMEM (Life Technologies, Inc., New York, NY) supplemented with 10% fetal bovine serum (JRH BIOSCIENCE, San Antonio, TX), penicillin, streptomycin (Life Technologies, Inc.), and amphotericin B (Fungizone; Life Technologies, Inc.).

Neutral lipids and casein were stained after induction of cell differentiation with 1.6 nm TPA (Funakoshi, Tokyo, Japan;
15
). Neutral lipids were stained by a modified “Oil Red O in propylene glycol” method
(16)
, and casein was stained by the immunofluorescence method using sheep antibovine casein antibody (Biogenesis, Poole, United Kingdom) and FITC-conjugated antisheep IgG antibody (Organon Tekinika, West Chester, PA; Ref.
17
). A rat embryonic fibroblast cell line transfected with the lacI gene, BBR-2 (Stratagene), was used as a negative control for staining. The transplantability of the established carcinoma cell lines was tested by injecting 7–10 × 106 cells into the back of nude mice (CLEA Japan, Tokyo, Japan) and observing tumors macroscopically and microscopically after 5–7 weeks.

Analysis of lacI MnFs and Corrected MnFs.

Cells (102) in their log phase were plated in a well of a 96-well plate and were transferred serially to a well of a 12-well plate, to a 10-cm dish, and then to a 15-cm dish. When the cells were subconfluent, they were collected, and the final cell count was measured. High Mr genomic DNA was extracted by the phenol/chloroform extraction method
(19)
. The number of cell divisions observed in each culture was calculated by subtracting the initial cell number (102) from the final cell count. Plating efficiency was monitored in the initial plating and the transfer steps, and doubling time was measured by seeding 3 × 105 cells and counting their growth at 24, 48, 72, and 96 h.

λ phages were packaged from the genomic DNA using Transpack (Stratagene, La Jolla, CA), and lacI mutant phages were identified by plating infected Escherichia coli SCS-8 on a plate with 0.7 mg/ml of 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside
(14, 20)
. The mutant frequency was calculated as the number of mutant blue plaques per 106 plaques. For each experiment, at least 105 λ phage plaques were analyzed. MnF was calculated as the number of independent mutants, defined as mutants with different lacI sequences, per 106 plaques. Corrected MnF was defined as MnF obtained by analysis of DNA obtained from cell population that had undergone 107 cell divisions, and it was calculated as:

Analysis of hprt MRs.

Cells (102) in their log phase were plated in a well of a 96-well plate, and transferred serially to a well of a 12-well plate and to a 10-cm dish. After measuring the final cell count, all of the cells were plated at ∼104 cells per well in a 96-well plate. Twenty-four h after the plating, 6-thioguanine (Sigma Chemical Co., St. Louis, MO) was added to the medium at a concentration of 1.5 μg/ml, and hprt mutants were detected 6–8 weeks later
(5)
. For each cell line, 24 replicate experiments were performed. The MR was calculated based on the equation
(8)
of Luria and Delbrück
(21)
:
where r stands for the average number of mutants, Nt for the final cell count, and C for the number of replicate experiments. The hprt MnF was calculated as:

PCR-SSCP Analysis, Sequencing, and Microsatellite Mutation Analysis.

The lacI mutation was determined by PCR-SSCP analysis followed by direct sequencing
(22)
, using the DNA from a mutant λ phage as the template. The hprt mutation was determined by the reverse transcription-PCR method using the cDNA, synthesized from the mutant colonies by the Cells-to-cDNA kit (Ambion Inc., Austin, TX), and reported primers
(23)
. p53 mutations were screened by the PCR-SSCP method using two running conditions
(24)
. The microsatellite mutations were examined for microsatellite markers D1Rat27, D2Rat88, D3Rat63, D4Rat133, D5Rat10, D6Rat4, D7Rat107, D8Rat21, D9Rat3, D10Rat9, D11Mgh2, D12Mgh5, D13Mgh4, D14Mit3, D15Mgh4, D16Rat18, D17Rat99, D18 Mgh3, D19Rat11, D20 Mit1, and DXRat20. All of the primers were purchased from Research Genetics (Huntsville, AL). PCR was performed in the presence of [α-32P]dCTP, and the diluted samples were run in a 5% acrylamide gel without glycerol.

Analysis of lacI and hprt Mutation Types and Statistical Analysis.

lacI and hprt mutations were classified into three categories, base substitutions, single bp deletions, and other mutations
(14)
. The “other mutations” category included deletions of two or more bp, insertions, and other complex mutations. When a bp deletion mutation occurred in a consecutive run of the same nucleotide, the nucleotide with the smallest number was assigned as the mutation site. The difference in corrected MnFs was analyzed by the Student t test. The difference in the frequencies of mutational types was analyzed by the χ2 test.

RESULTS

Establishment of Mammary Carcinoma Cell Lines.

Two cell lines, PhIP12–1 and PhIP7–4, were established from two mammary carcinomas induced by PhIP. The primary carcinomas, from which PhIP12–1 and PhIP7–4 were derived, were histologically diagnosed as poorly differentiated adenocarcinoma and moderately differentiated adenocarcinoma, respectively. The morphology of PhIP12–1 and PhIP7–4 cell lines is shown in Fig. 1, A and B⇓
. To confirm their origin from the mammary epithelium, their production of lipid droplets and casein were examined. Lipid droplets with a granulated pattern were observed in these two carcinoma cell lines (E and F), whereas no granulation was observed in a control fibroblast cell line (H). Casein production was observed in the cytoplasm (I and J), whereas only a nonspecific background was present in the control (L). Karyotype analysis showed that the PhIP12–1 cell line was with hyperploidy (chromosome number, 69–102) and its chromosome number was highly variable (data not shown). On the other hand, the PhIP7–4 cell line showed a normal karyotype. The two cell lines were confirmed to form adenocarcinomas in two independent transplantability experiments.

Morphology (A–D; ×100), lipid staining (E–H; ×250), and casein staining (I–L; ×100) of PhIP12-1 (A, E, and I), PhIP7-4 (B, F, and J), primary culture of the normal mammary epithelium (C, G, and K), and BBR-2 fibroblast (D, H, and L). Lipid staining and casein staining were performed after induction of cell differentiation with 1.6 nm of TPA for 3 days. BBR-2 fibroblast was used as a negative control to exclude nonspecific staining.

Increase of lacI-corrected MnFs in the Carcinoma Cell Lines.

To obtain corrected MnFs for the lacI gene, which reflect the number of spontaneous mutations that occurred during a culture from 102 cells to 107 cells (see “Materials and Methods”), six independent cultures were performed for each carcinoma cell line, and ten cultures were performed for the primary culture of the normal mammary epithelium (Table 1)
⇓
. The mammary origin of the primary culture was confirmed by lipid and casein production (Fig. 1, G and K)
⇓
. The plating efficiencies in the initial plating and two transfers were 94 ± 1.5%, 89 ± 4.4%, and 81 ± 5.2% (mean ± SE) for PhIP12–1, PhIP7–4, and the control primary culture of the normal mammary epithelium, respectively. The average doubling times were 22 ± 0.32 h, 25 ± 0.24 h, and 32 ± 1.4 h, respectively.

lacI MnFs and corrected MnFs in the primary culture of normal mammary epithelium and two mammary carcinomas

After sequencing all of the lacI mutants, the corrected MnFs of PhIP12–1 and PhIP7–4 were calculated as 59 ± 8.6 × 10−6 and 72 ± 4.1 × 10−6 (mean ± SE), respectively, whereas that of the normal mammary epithelium was 4.7 ± 1.6 × 10−6. The corrected MnFs in the carcinoma cell lines were significantly increased compared with that in the normal mammary epithelium (P < 10−3 and P < 10−6 for PhIP12–1 and PhIP7–4, respectively).

Increase of hprt MRs in the Carcinoma Cell Lines.

Spontaneous MRs of the endogenous hprt gene were measured in 24 replicate experiments for each of the two carcinoma cell lines and the control (Table 2)
⇓
. The hprt MRs of PhIP12–1 and PhIP7–4 were calculated as 8.2 × 10−7 and 11 × 10−7 mutations/cell division/hprt, respectively, whereas that of the normal mammary epithelium was 1.4 × 10−7. MnFs of the hprt gene, which reflects the number of spontaneous mutations that occurred during a culture from 102 cells to 106 cells, were 2.8 ± 0.28 × 10−6 and 3.9 ± 0.45 × 10−6 (mean ± SE) per cell for PhIP12–1 and PhIP7–4, respectively, whereas that of the normal mammary epithelium was 0.17 ± 0.08 × 10−6. The increases of corrected MnFs in the carcinoma cell lines were statistically significant with Ps less than 10−9 and 10−10, respectively.

hprt MRs and MnFs in the primary culture of normal mammary epithelium and two mammary carcinoma cell lines

Mutational Types in the Carcinoma Cell Lines and Primary Carcinomas.

Analysis of the mutational types of the lacI gene (Table 3)
⇓
revealed that both the PhIP12–1 and the PhIP7–4 cell lines showed high frequencies of all of the mutations compared with the primary culture of the normal mammary epithelium cells. In particular, the high frequencies of A:T to C:G (25% for PhIP12–1 and 24% for PhIP7–4) transversion were in clear contrast with the absence of A:T to C:G transversion in the primary culture of the normal mammary epithelium (0 of 6) and in the in vivo normal mammary tissue (0 of 34; Ref.
14
). A:T to C:G transversion at nucleotide 369 was detected in 8 of 12 experiments performed in the two carcinoma cell lines, whereas it was absent in the primary culture of the normal mammary epithelium and in the normal mammary tissue (Table 4
⇓
; Ref.
14
). A:T to C:G transversion at nucleotide 369 was thus considered as a hotspot mutation in the carcinoma cell lines.

Analysis of the mutational types of the hprt gene confirmed the same tendency. Both the PhIP12–1 and the PhIP7–4 cell lines showed higher frequencies of almost all of the mutations than did the control, and the high frequency of A:T to C:G (15 and 21%, respectively) transversion in the carcinoma cell lines was in clear contrast to the absence of A:T to C:G (0 of 4; Table 3
⇓
). Detailed hprt mutations are listed in Table 5
⇓
.

lacI mutational types were analyzed in the primary carcinomas. A total of 71 different mutations were identified from 124 mutants in the primary carcinomas 12-1 and 7-4. The MnFs in them were 36 × 10−6 and 53 × 10−6, respectively (Table 1)
⇓
. The A:T to C:G transversion constituted ∼20% of the total mutations (Table 3)
⇓
, and the mutation at nucleotide 369 was observed in both of the primary carcinomas at multiple times (Table 4)
⇓
. G:C deletion, which is known to be characteristic of PhIP-induced mutations
(14, 25)
and was absent in the carcinoma cell lines, was observed at 5–9% in the primary carcinomas (Table 3)
⇓
.

Absence of Microsatellite Mutations and p53 Mutations.

To examine whether or not microsatellite instability was present, 21 loci microsatellites were analyzed using DNA obtained from each of the six cultures of PhIP12–1 and PhIP7–4 and of primary carcinomas 12-1, and 7-4. No alterations in the lengths of the microsatellites were detected in any of 21 loci in any of the DNA samples.

Exons 2 through 9 of the p53 gene, covering codons 1 through 365 and all of the exon-intron junctions, were analyzed by PCR-SSCP analysis using the same DNA samples. However, no shifted bands were observed in any region of any of the samples.

DISCUSSION

Two rat mammary carcinoma cell lines were established. The two cell lines demonstrated 12- and 15-fold increases in the corrected MnFs of the lacI marker gene, 6- and 8-fold increases in the MRs of the endogenous hprt gene, and 16- and 23-fold increases in the MnFs of the hprt gene, compared with the primary culture of normal mammary epithelium. The corrected MnFs in this study reflect the dynamic increase of point mutations, and it can be concluded that both of the two cell lines had increased rates of spontaneous point mutations using two different marker genes. The spontaneous MR in mammalian cells shows a highly consistent value, 1–2 × 10−7 mutations/cell division/hprt, in many studies
(2)
, and the value was reproduced in this study. Because the spontaneous MR is not sufficient to induce multiple mutations in a single cell
(1, 2, 26)
, the 6- to 8-fold increases of the MRs in the carcinoma cell lines are expected to result in a significant increase in the chance of developing mammary carcinomas. Among the three parameters measured, the hprt MnFs showed the largest increase, followed by the lacI MnF, and then by the hprt MR. For a range of MR and final cell count, the equation
(8)
of Luria and Delbrück
(21)
is known to give a larger increase in the average numbers of mutants, reflected in the MnFs, than that in the MRs. The larger increase of the hprt MnF than that of the lacI MnF was considered to be attributable to a difference in the target genes.

The mammary carcinoma cell lines established in this study, along with their primary carcinomas, lacked microsatellite instability. Therefore, the increase in the spontaneous point MRs found here can be distinguished from microsatellite instability, which is generally associated with increased rates of point mutations
(5,
6,
7,
8)
. We would term this category of gnomic instability as SNI, and this finding expands the original observation by Eshleman et al. in a colon cancer cell line
(5, 9)
to mammary carcinoma cell lines.

Both the lacI and the hprt genes showed essentially the same mutation types. The increase of A:T to C:G transversion was characteristic (6 of 24 and 6 of 25 for lacI; 10 of 67 and 19 of 92 for hprt), compared with the control (0 of 6 for lacI; 0 of 4 for hprt). This type of mutation is known to be infrequent in any untreated or treated tissues of BBR.
4
At nucleotide 369 of the lacI gene, a clustering of mutations was observed in the two carcinoma cell lines, whereas no clustering of spontaneous or induced mutations had been reported previously. Both the high frequency of A:T to C:G transversion and the clustering of mutations at nucleotide 369 were present in the primary carcinomas, in which the analysis of MRs is impossible, and it was strongly indicated that the SNI was also present in the primary carcinomas. On the basis of a wide range of models on the role of genetic instability in carcinogenesis
(3)
, we speculate that SNI was induced in the early stage of carcinogenesis, probably by the action of PhIP, and then spontaneous mutations in tumor-related genes were accumulated to yield a mammary tumor.

The responsible mechanism for the SNI could be diverse. The SNI in this report had a preference for A:T to C:G transversions and probably for the sequence context around nucleotide 369. The colon cancer cell line that displayed SNI also showed a high frequency of A:T to C:G transversions (5 of 32;
9
). One possible mechanism is the inactivation of the mutT gene. MutT product removes oxo-dGTP from the nucleotide pool, and its inactivation induces a high frequency of A:T to C:G transversion in E. coli(27, 28)
. Another possible mechanism is the decreased fidelity of DNA polymerases, which has been expected to lead to this type of SNI
(29, 30)
. Because PhIP is known to form DNA adduct mainly at C8 position guanine bases
(31)
, there is a good chance that a key gene for SNI was mutated at a G base and the SNI was established.

Some previous reports using marker transgenic animals showed higher mutant frequencies in their tumors
(32)
. There is also a report that showed a mutant frequency in tumors similar to that in the normal mammary epithelium
(33)
. MnF in a tumor is determined by the number of mutations that occurred at any time between the completion of the initial tumor cell and the sampling of the tumor. MnF in a normal tissue is determined by the number of mutations that occurred at any time between the completion of the embryonic development of the tissue and the sampling of the tissue. For direct comparison of the dynamic rate of occurrence of point mutations, establishment of cell lines is requisite, and this is the first report in which cell lines were established from tumors induced in marker transgenic animals.

No microsatellite mutations were observed in the 21 microsatellite loci of any of the DNA samples, and we can conclude that microsatellite instability was lacking in the cell lines and primary carcinomas. When microsatellite instability is present in tumors or cell lines, microsatellite mutations are known to be observed at rates of 7–95%
(34)
. Tumors that developed in Msh2-deficient mice also displayed a high rate of microsatellite mutations
(35)
. p53 mutation is also involved in genomic instability but is known to cause no significant increase in point mutations
(36)
. As expected, no p53 mutations were detected in this study. Therefore, the SNI observed in this study was not attributable to mechanisms that would cause microsatellite instability, nor to p53 mutations.

Plating efficiencies were within the same range for the carcinoma cell lines and the primary culture. All of the cells were kept in log-phase throughout the experiments, and recognizable cell death was absent in microscopic observations. Even if we had neglected preferential occurrence of cell death in the primary culture of normal mammary epithelium, this would have led to underestimation of the underlying number of these cell divisions and to overestimation of the corrected MnF. Seeding 102 cells enabled us to confirm that no lacI mutant was present at the start of the experiments. If a single mutant had existed at the start of an experiment, it could have been detected as an extraordinarily high mutant frequency with a clonal origin. The final mutant frequency should be 250 × 10−6 (1/100/40), because there are 40 copies of the lacI gene in a cell.

Now, it is suggested that SNI is not an exceptional category of genomic instability restricted to a colon cancer cell line. We need additional studies on the mechanism responsible for SNI, the timing of its occurrence, and the range of tumors with SNI.

Acknowledgments

We thank Dr. Y. Kuroda for his kind advice and K. Wakazono and Dr. M. Nakayasu for their technical assistance.

Footnotes

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

↵1 Supported by a Grant-in-Aid for Cancer Research, by a Grant-in-Aid for the 2nd term Comprehensive 10-Year Strategy for Cancer Control from the Ministry of Health and Welfare of Japan, and by a grant from the Nishi Memorial Foundation.